Canine transient receptor potential v2 (ctrpv2) and methods of screening for trpv2 channel modulators

ABSTRACT

A recombinant canine TRPV2 channel which has been prepared by cDNA cloning and polymerase chain reaction techniques is disclosed. Expression systems for these channels and an assay using the expression systems are also disclosed. The recombinant TRPV2 channel can be used in assays to evaluate compounds which directly or indirectly interact with or bind to TRPV2 channel.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to Application No. 60/893,445 filed onMar. 7, 2007, the entire contents of which are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to the canine TRPV2 (cTRPV2) channel,cloning of the channel, methods of screening for modulators of the TRPV2channel and uses thereof.

BACKGROUND OF THE INVENTION

Both heat and cold evoke thermosensation, which, if of sufficientintensity, may elicit feelings of pain. Temperature detection in mammalsis a critical function in the maintenance of thermal homeostasis andprotection from potentially injurious temperature extremes. Considerableefforts have been put into elucidating the biochemical mechanismsinvolved in the detection, transduction and transmission of hot and coldsensations in neuronal tissues. Thermal stimuli activate specializedreceptors located on sensory neurons, such as those deriving from thedorsal root ganglion (DRG) and the trigeminal ganglion (TG). When thesestimuli are in the noxious range (i.e., very hot or cold), they activatea certain subset of thermo-sensitive receptors on a sub-population ofsensory neurons called nociceptors (pain-sensing neurons). Uponactivation, the thermo-sensitive receptors (e.g., ion channels)transduce the noxious stimulus into an electrical signal that ispropagated along the sensory neuron to the spinal cord, where it isrelayed to the brain, ultimately leading to the perception of pain.Accordingly, these thermo-sensitive receptors represent highly promisingtargets for developing drugs for the treatment of various painfulconditions, particularly those in which thermal hypersensitivity and/orthermally evoked pain are present.

Several thermo-sensitive receptors have been implicated in sensingtemperature changes. Currently, six members from the transient receptorpotential (TRP) ion channel family have been proposed to serve asthermal sensors of temperatures ranging from noxious heat to noxiouscold and thus are referred to as thermo-TRPs. The thermo-TRPs, which arenon-selective cation channels, are also sensitive to chemical stimuli,such as capsaicin, menthol mustard oil, etc., which are known to producea burning or cooling sensation. Importantly, all of the thermo-TRPs areexpressed in nociceptive sensory neurons, either Aδ- or C-fibers,wherein thermo-, mechano- and chemo-sensory transduction take place.TRPV1, the most extensively characterized member of this family, isactivated by moderate heat (˜43° C.), capsaicin, protons and certainlipidic mediators, such as anandamide (Caterina, M. J., et al. 1997,Nature 389 (6653), 816-824; Tominaga, et al. 1998, Neuron 21(3),531-543) Moreover, numerous studies using a variety of experimentalapproaches have implicated TRPV1 in the transduction of pain signals inanimal models of hyperalgesia (Caterina, M. J., et al. 2000, Science 288(5464), 306-313; Davis, J. B., et al. 2000 Nature 405 (6783), 183-187);hence, this TRP has been aggressively pursued as a target for thealleviation of certain types of pain.

The transient receptor potential V2 (TRPV2) protein (previously calledand also known as vanilloid receptor-like 1 or VRL-1) is a member of thetransient receptor potential (TRP) superfamily, which has beenimplicated in mediating diverse cellular functions, including thetransduction of thermal, chemical and mechanical stimuli. TRPV2 isclosely related to TRPV1 and was identified by homologous cloning fromrat and human (Caterina et al., 1999, Nature 398 (6726), 436-441) aswell as mouse (Kanzaki, et al. 1999, Nat Cell Biol 1 (3), 165-170).Functional studies have revealed that rat TRPV2 responds to noxiousheat, with an activation threshold above 52° C. (Caterina, Rosen et al.1999) and that mouse TRPV2 responds to changes in osmolarity or tomembrane stretch (Muraki, et al. 2003, Circ Res 93 (9), 829-838). Thehypothesis that TRPV2 is an endogenous sensor of noxious heat andmechanical stretch is further supported by findings that it is expressedin medium to large diameter Aδ mechano- and thermo-sensory neurons inthe dorsal root ganglion (DRG) (Caterina et al., 1999, Nature 398(6726), 436-441).

Unlike TRPV1, however, no selective activators of TRPV2 have beenreported. Recently, a novel class of selective agonists, such asΔ⁹-tetrahydrocannabinol (Δ⁹-THC) as well as other related cannabinoids,that activate human, rat and mouse TRPV2 have been identified. (See U.S.application Ser. No. 11/589,340, entitled COMPOSITIONS AND METHODS FORIDENTIFYING MODULATORS OF TRPV2 to Qin et al., filed Oct. 30, 2006).

There is a need to identify additional thermo-sensitive receptors, asthey are potential targets for the treatment of pain. There is also aneed to identify thermo-sensitive receptors in different species, asthey can be used as model systems to investigate the effects of testcompounds. Particularly, there is a need for systems that can be used totest compounds that potentially increase or decrease the activity of athermo-sensitive receptor responding to noxious thermal stimuli,including noxious heat. Identification and testing of such compoundswould enable the treatment of various disorders associated withneuropathic pain, inflammatory pain, various chronic pains or for usesin other conditions, such as the inflammatory immune response, febrileseizures and general epilepsy and/or conditions in which thermalhypersensitivity or thermally evoked pain are present as well as otherhuman diseases related to these thermo-sensitive receptors.

SUMMARY OF THE INVENTION

The present invention is directed to recombinant DNA and protein ofcanine TRPV2. The protein, which may be used in a high throughput assay,is useful to identify modulators of functional TRPV2. The presentinvention is also directed to modulators identified in the assaysdisclosed herein and their use as therapeutic agents.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the nucleotide sequence and protein translation ofcanine TRPV2 cDNA (SEQ ID NO: 1).

FIG. 2 illustrates the amino acid sequence of canine TRPV2 (SEQ ID NO:2).

FIG. 3 illustrates an alignment of the canine TRPV2 nucleotide sequenceand the human TRPV2 nucleotide sequence (Genbank accession #: AF129112)(SEQ ID NO: 3). The sequences share 87.0 percent similarity and 84.2percent identity.

FIG. 4 illustrates an alignment of the canine TRPV2 amino acid sequenceand the human TRPV2 amino acid sequence (Genbank accession #: AF129112)(SEQ ID NO: 4). The sequences share 85.9 percent similarity and 85.9percent identity.

FIG. 5 illustrates the percent homology between human, murine, rat andcanine TRPV2 peptide sequences.

FIG. 6 illustrates the change in cellular calcium levels with additionof 100 μM Δ⁹-tetrahydrocannabinol (THC) in HEK293 cellstransiently-transfected with canine TRPV2-expressing (black line) orpcDNA3.1 vector control (gray line) plasmid DNA. RFU; RelativeFluorescent Units.

DETAILED DESCRIPTION OF THE INVENTION

All publications cited herein are hereby incorporated by reference.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. As used herein, the terms“comprising”, “containing”, “having” and “including” are used in theiropen, non-limiting sense.

“An activity”, “a biological activity”, or “a functional activity” of apolypeptide or nucleic acid refers to an activity exerted by apolypeptide or nucleic acid molecule as determined in vivo or in vitro,according to standard techniques. Such activities can be a directactivity, such as an association with or an enzymatic activity on asecond protein or an ion enzyme activity, or an indirect activity, suchas a cellular signaling activity mediated by interaction of the proteinwith one or more than one additional protein or other molecule(s),including, but not limited to, interactions that occur in a multi-step,serial fashion.

A “biological sample” or “sample” as used herein refers to a samplecontaining or consisting of cell or tissue matter, such as cells, cellassociated body fluids, biological fluids, culture supernatants, DNA,RNA, or protein isolated from a subject or patient. The “subject” can bebacteria, yeast, arthropods, a mammal, such as a rat, a mouse, a canine,a human, or any other organism, that has been the object of treatment,observation or experiment. Examples of biological samples include, forexample, saliva, sputum, blood, blood cells (e.g., white blood cells),amniotic fluid, plasma, semen, bone marrow, tissue or fine-needle biopsysamples, urine, peritoneal fluid, pleural fluid, and cell cultures.Biological samples can also include sections of tissues, such as frozensections taken for histological purposes.

A “cell” refers to at least one cell or a plurality of cells appropriatefor the sensitivity of the detection method. Cells suitable for thepresent invention can be mammalian, for example, canine cells, or otherprokaryote cells.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis. A “cell line” is derived from clonal expansion ofprimary cells, and is capable of stable growth in vitro for manygenerations.

A “DNA clone” is a section of DNA that has been copied, isolated orremoved from a host and inserted into a vector molecule, such as aplasmid or a phage, or a chromosome, and then replicated to form manyidentical copies.

The term “cDNA” as used herein means a complementary DNA (cDNA) that hasbeen reverse transcribed from a specific RNA template through the actionof the enzyme reverse transcriptase.

A “gene” is a segment of DNA involved in producing a peptide,polypeptide, or protein, and the mRNA encoding such protein species,including the coding region, non-coding regions preceding (“5′UTR”) andfollowing (“3′UTR”) the coding region. A “gene” can also includeintervening non-coding sequences (“introns”) between individual codingsegments (“exons”). “Promoter” means a regulatory sequence of DNA thatis involved in the binding of RNA polymerase to initiate transcriptionof a gene. Promoters are often upstream of (or 5′ to) the transcriptioninitiation site of the gene. A “regulatory sequence” refers to theportion of a gene that can control the expression of the gene. A“regulatory sequence” can include promoters, enhancers and otherexpression control elements such as polyadenylation signals, ribosomebinding site (for bacterial expression), and/or an operator.

“Nucleic acid sequence” or “nucleotide sequence” refers to thearrangement of either deoxyribonucleotide or ribonucleotide residues ina polymer in either single- or double-stranded form. Nucleic acidsequences can be composed of natural nucleotides of the following bases:thymidine, adenine, cytosine, guanine, and uracil; abbreviated T, A, C,G, and U, respectively, and/or synthetic analogs. Synthetic analogs caninclude nucleotide and nucleoside analogs as well as non-nucleotide andnon-nucleoside analogs.

The term “oligonucleotide” refers to a single-stranded DNA or RNAsequence of a relatively short length, for example, less than 100residues long. For many methods, oligonucleotides of about 15-40nucleotides in length are useful, although longer oligonucleotides ofgreater than about 40 nucleotides can sometimes be utilized. Someoligonucleotides can be used as “primers” for the synthesis ofcomplimentary nucleic acid strands. For example, DNA primers canhybridize to a complementary nucleic acid sequence to prime thesynthesis of a complementary DNA strand in reactions using DNApolymerases. Oligonucleotides are also useful for hybridization inseveral methods of nucleic acid detection, for example, in Northernblotting or in situ hybridization.

The term “probe” as used herein refers to a nucleic acid that canselectively hybridize under stringent hybridization conditions to anucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide capable of TRPV2 activity and having at least 90% sequenceidentity to SEQ ID NO: 2. Additionally, a probe can selectivelyhybridize under stringent hybridization conditions to a polypeptidecapable of TRPV2 activity and having at least 90% sequence identity toSEQ ID NO: 2. Nucleic acid probes can be of any practical length similarto oligonucleotides in general and can be RNA or DNA, as describedabove. For example, probes can be from 10-20, 21-40, 41-60, or greaterthan 61 nucleotides in length. Such probes are useful for the detectionof TRPV2 molecules in heterogeneous mixtures or for the amplification ofrare copies, genomic copies and recombinant copies in vectors, as in thecase of polymerase chain reactions, South-western blots and other commonmolecular biology techniques. Hybridization conditions are well known inthe art, for example, but not limited to, those described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., (1989).

A “polypeptide sequence” or “protein sequence” refers to the arrangementof amino acid residues in a polymer. Polypeptide sequences can becomposed of the standard 20 naturally occurring amino acids, in additionto rare amino acids and synthetic amino acid analogs. Shorterpolypeptides are generally referred to as peptides.

An “isolated” or “purified” nucleic acid molecule is one that issubstantially separated from other nucleic acid molecules present in thenatural source of the nucleic acid. An “isolated” nucleic acid moleculecan be, for example, a nucleic acid molecule that is free of at leastone of the nucleotide sequences that naturally flank the nucleic acidmolecule at its 5′ and 3′ ends in the genomic DNA of the organism fromwhich the nucleic acid is derived. Isolated nucleic acid moleculesinclude, without limitation, separate nucleic acid molecules (e.g., cDNAor genomic DNA fragments produced by PCR or restriction endonucleasetreatment) substantially independent of other sequences, as well asnucleic acid molecules that are incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid molecule can include anucleic acid molecule that is part of a hybrid or fusion nucleic acidmolecule. An isolated nucleic acid molecule can be a nucleic acidmolecule that is: (i) amplified in vitro by, for example, polymerasechain reaction (PCR); (ii) synthesized by, for example, chemicalsynthesis; (iii) recombinantly produced by cloning; or (iv) purified,for example, by cleavage and electrophoretic or chromatographicseparation.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium (i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation). When the proteinis produced by chemical synthesis, it is preferably substantially freeof chemical precursors or other chemicals (i.e., it is separated fromchemical precursors or other chemicals that are involved in thesynthesis of the protein). Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, and 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest. Isolatedbiologically active polypeptides can have several different physicalforms. An isolated polypeptide can exist as a full-length nascent orunprocessed polypeptide, or as a partially processed polypeptide or as acombination of processed polypeptides. The full-length nascentpolypeptide can be posttranslationally modified by specific proteolyticcleavage events that result in the formation of fragments of thefull-length nascent polypeptide. The full-length protein or fragments ofthe polypeptide can be chemically modified. A fragment, or physicalassociation of fragments can have the biological activity associatedwith the full-length polypeptide; however, the degree of biologicalactivity associated with individual fragments can vary. An isolated orsubstantially purified polypeptide, can be a polypeptide encoded by anisolated nucleic acid sequence as well as a polypeptide synthesized by,for example, chemical synthetic methods, and a polypeptide separatedfrom biological materials, and then purified, using conventional proteinanalytical or preparatory procedures, to an extent that permits it to beused according to the methods described herein.

The terms “reducing expression”, “decreasing expression”, “knockingdown” and “knock down” as used herein to refer to the reduction in theexpression of one or more than one gene. This reduction in geneexpression can be as a result of a reduction in the rates oftranscription or translation or an increase in the rates of degradationor any combination of the above. For example, TRPV2 gene expression canbe reduced through the use of RNA interference technology. Suitable RNAinterference techniques are described and reviewed by Sandy et. al inBiotechniques. 2005 August; 39 (2): 215-24, Mammalian RNAi: a PracticalGuide, which is incorporated by reference.

The term “antisense” as used herein refers to a noncoding molecule thatis complementary to the bases of a coding sequence of mRNA. Introducinga transgene coding for antisense mRNA is another strategy used to reduceor knock down expression of a gene. A strand of antisense mRNA can alsobe introduced into the cytosol by microinjection.

Analogous molecules with modified backbones have been designed, whichchange various characteristics of RNA, such as its instability todegradative enzymes, thermal stability and bond strength, therebyaltering gene expression. Some alternative antisense structural typesinclude phosphorothioate, PNA (peptide nucleic acid), LNA (lockednucleic acid), and 2′-O alkyl oligos.

The term “selectively hybridize” as used herein refers to the ability ofa probe to bind to a particular or intended nucleic acid or polypeptidetarget sequence or molecule in a heterogeneous mixture of nucleic acidsor polypeptides, for example, a probe can selectively hybridize to anucleotide sequence encoding a TRPV2 or a polypeptide capable of TRPV2activity in a heterogeneous mixture.

“Recombinant” refers to a nucleic acid, a protein encoded by a nucleicacid, a cell, or a viral particle, that has been modified usingmolecular biology techniques to something other than its natural state.For example, recombinant cells can contain nucleotide sequence that isnot found within the native (non-recombinant) form of the cell or canexpress native genes that are otherwise abnormally expressed,under-expressed, or not expressed at all. Recombinant cells can alsocontain genes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termalso encompasses cells that contain an endogenous nucleic acid moleculethat has been modified without removing the nucleic acid from the cell;such modifications include those obtained, for example, by genereplacement and site-specific mutation.

A “recombinant host cell” is a cell that has had introduced into it arecombinant DNA sequence. Recombinant DNA sequences can be introducedinto host cells using any suitable method, including, for example,electroporation, calcium phosphate precipitation, microinjection,transformation, biolistics “gene-gun” and viral infection. RecombinantDNA may or may not be integrated (covalently linked) into chromosomalDNA making up the genome of the cell. For example, the recombinant DNAcan be maintained on an episomal element, such as a plasmid.Alternatively, with respect to a stably transformed or transfected cell,the recombinant DNA has become integrated into the chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the stably transformed ortransfected cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA. Recombinanthost cells can be prokaryotic or eukaryotic, including, E. coli, fungalcells, such as yeast, mammalian cells, such as cell lines of human,bovine, porcine, canine and rodent origin, and insect cells, such asDrosophila- and silkworm-derived cell lines. It is further understoodthat the term “recombinant host cell” refers not only to the particularsubject cell but also to the progeny or potential progeny of such acell. Because certain modifications can occur in succeeding generations,due to either mutation or environmental influences, such progeny cannot,in fact, be identical to the parent cell but are still included withinthe scope of the term as used herein.

The term “vector” or “construct” refers to a nucleic acid molecule intowhich a heterologous nucleic acid can be or is inserted. Some vectorscan be introduced into a host cell allowing for replication of thevector or for expression of a protein that is encoded by the vector orconstruct. Vectors typically have selectable markers, for example, genesthat encode proteins allowing for drug resistance, origins ofreplication sequences, and multiple cloning sites that allow forinsertion of a heterologous sequence. Vectors are typicallyplasmid-based and are designated by a lower case “p” followed by acombination of letters and/or numbers. Starting plasmids disclosedherein are either commercially available, publicly available on anunrestricted basis, or can be constructed from available plasmids byapplication of procedures known in the art. Many plasmids and othercloning and expression vectors that can be used in accordance with thepresent invention are well known and readily available to those of skillin the art. Moreover, those of skill readily can construct any number ofother plasmids suitable for use in the invention. The properties,construction and use of such plasmids, as well as other vectors, in thepresent invention will be readily apparent to those of skill from thepresent disclosure.

The term “expression” as used herein refers to a multi-step process thatincludes transcription and translation of a gene and is often followedby post-translational modification, folding, assembly andtrafficking/targeting of the resulting protein. The amount of proteinthat a cell expresses depends on the tissue, the developmental stage ofthe organism and the metabolic or physiologic state of the cell.

The term “expression vector” refers to a vector that comprisesregulatory sequences necessary for transcription and translation of acloned gene or genes and thus can transcribe and clone DNA. Anexpression vector can include one or more than one regulatory sequence,which can be selected based on the type of host cells used, operablylinked to a gene. Regulatory sequences include promoters, enhancers andother expression control elements, for example, poly (A)+ sequences.Other expression vector components can include, but are not limited to,one or more than one of the following: a signal sequence, an origin ofreplication, one or more than one selection gene and a transcriptiontermination sequence.

“Sequence” means the linear order in which monomers occur in a polymer,for example, the order of amino acids in a polypeptide or the order ofnucleotides in a polynucleotide.

“Sequence identity or similarity”, as known in the art, is therelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Asused herein, “identity”, in the context of the relationship between twoor more nucleic acid sequences or two or more polypeptide sequences,refers to the percentage of nucleotide or amino acid residues,respectively, that are the same when the sequences are optimally alignedand analyzed. For the purposes of comparing a queried sequence against,for example, the amino acid sequence SEQ ID NO: 2, the queried sequenceis optimally aligned with SEQ ID NO: 2 and the best local alignment overthe entire length of SEQ ID NO: 2 (331 amino acids) is obtained.

Analysis can be carried out manually or using sequence comparisonalgorithms. For sequence comparison, typically one sequence acts as areference sequence, to which a queried sequence is compared. When usinga sequence comparison algorithm, test and reference sequences are inputinto a computer, sub-sequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated.

Optimal alignment of sequences for comparison can be conducted, forexample, by using the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol., 48:443 (1970). Software for performing Needleman& Wunsch analyses is publicly available through the Institut Pasteur(France) Biological Software website. The NEEDLE program uses theNeedleman-Wunsch global alignment algorithm to find the optimumalignment (including gaps) of two sequences when considering theirentire length. The identity is calculated along with the percentage ofidentical matches between the two sequences over the reported alignedregion, including any gaps in the length. Similarity scores are alsoprovided wherein the similarity is calculated as the percentage ofmatches between the two sequences over the reported aligned region,including any gaps in the length. Standard comparisons utilize theEBLOSUM62 matrix for protein sequences and the EDNAFULL matrix fornucleotide sequences. The gap open penalty is the score taken away whena gap is created; the default setting using the gap open penalty is10.0. For gap extension, a penalty is added to the standard gap penaltyfor each base or residue in the gap; the default setting is 0.5.

Hybridization can also be used as a test to indicate that twopolynucleotides are substantially identical to each other.Polynucleotides that share a high degree of identity will hybridize toeach other under stringent hybridization conditions. “Stringenthybridization conditions” has the meaning known in the art, as describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory, Cold. Spring Harbor, N.Y.,(1989). An exemplary stringent hybridization condition for anoligonucleotide probe comprises hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morethan one wash in 0.2× SSC and 0.1% SDS at 50- 65° C., depending upon thelength over which the hybridizing polynucleotides share complementarity.

A “reporter gene” refers to a nucleic acid sequence that encodes areporter gene product. As is known in the art, reporter gene productsare typically easily detectable by standard methods. Exemplary suitablereporter genes include, but are not limited to, genes encodingluciferase (lux), β-galactosidase (lacZ), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-glucuronidase,neomycin phosphotransferase, and guanine xanthinephosphoribosyl-transferase proteins.

The term “marker” as used herein refers to a specific substance from asubject that has a particular molecular feature that makes it useful formeasuring the progress of disease or the effects of treatment.Biological samples comprising the marker are taken at periodic intervalsfrom the subject to measure the progress of disease or the effects oftreatment. The first measure of the marker is compared to the secondmeasure and/or subsequent measures. In another example, the measure ofthe marker can be compared to a reference, a standard or a negativecontrol.

TRPV's are vanilloid sub-family members of the 6 transmembrane domaintransient receptor potential (TRP) superfamily. Structural, biochemicaland pharmacological analyses of the TRPV genes and proteins has led tothe discovery of 6 distinct members, including TRPV1 and TRPV2, whichare thermo-sensitive to noxious heat. Activation of TRPV's mediatesseveral receptor subtype-specific physiological processes that includebut are not limited to the regulation of cellular calcium levels,excitability and transmitter release as well as sensory transduction andhomeostasis.

The term “TRPV2 channel,” as used herein includes any receptor that canbe naturally occurring, cloned, mutated, recombinantly expressed,chemically modified or substituted with amino acid analogs whilemaintaining TRPV channel activity.

The term “epitope” as used herein refers to a site on a large moleculeagainst which an antibody may be produced and to which it will bind.Epitopes can be naturally occurring or chemically or syntheticallyproduced.

A “compound that decreases the activity of TRPV2 channel” includes anycompound that results in decreased signaling through the TRPV2 channel.In one embodiment, such a compound can be a partial agonist. In anotherembodiment, such a compound can be an antagonist, which can be eithercompetitive or non-competitive. In another embodiment the compound canbe both a partial agonist and an antagonist.

A “compound that increases the activity of TRPV2 receptor” includes anycompound that results in increased signaling through the TRPV2 channel.Such a compound can be an agonist or a partial agonist.

A kit is any manufacture (e.g., a package or container) comprising atleast one reagent, the manufacture being promoted, distributed, or soldas a unit for performing the methods of the present invention.

A putative homolog of TRPV2 from canine (SEQ ID NO: 1) has now beencloned. The TRPV2 of canine provides a means for identification of TRPV2antagonists and agonists with potential utility for treating certaintypes of pain and painful conditions, including but not limited toneuropathic pain and inflammatory pain. An alignment of the canine TRPV2nucleotide sequence and the human TRPV2 nucleotide sequence is shown inFIG. 3. FIG. 3 shows an 86 percent sequence identity between these twosequences. An alignment of the canine TRPV2 amino acid sequence and thehuman TRPV2 amino acid sequence is shown in FIG. 4. FIG. 4 shows an 84percent sequence identity between these two sequences.

The present invention relates to novel TRPV2 nucleic acids, polypeptidesand proteins encoded by these nucleic acids, recombinant TRPV2materials, and methods involving the production, detection, andutilization of these materials.

Inhibition of the function or expression of TRPV2 proteins can beadvantageous for screening and validating drug candidates both in vitroand in vivo. Since TRPV2 is implicated in noxious heat sensation, it isanticipated that inhibition of TRPV2 activity is also relevant fortherapeutic applications where neuropathic, inflammatory and chronicpains occur.

In attempts to clone the TRPV2 orthologous, a PCR-based strategy wasemployed. Oligonucleotide primers were synthesized according to thesequences set forth in SEQ ID NO: 5 and. SEQ ID NO: 6. These primerswere successfully used to amplify a portion of the TRPV2 sequence fromposition 761 to position 2200 SEQ ID NO: 1. The full length cDNA wasthen cloned by both 5′ and 3′ RACE-PCR. In the present invention, theTRPV2 gene was cloned from the cDNA of canine dorsal root ganglion. TheTRPV2 gene was sequenced, and the nucleotide sequence is shown as SEQ IDNO: 1 (see also, FIG. 1). SEQ ID NO: 1 encodes a 765 residue polypeptide(see, SEQ ID NO: 2), as shown in FIG. 2.

In the present invention, the TRPV2 nucleic acid was also subcloned intoan expression vector and transformed into a host cell for expression ofthe TRPV2 protein. This recombinant TRPV2 cell system was shown toexpress a functional TRPV2 protein that responded to Δ⁹-THC in adose-dependent manner.

In other diagnostic assays which are capable of detecting the expressionof TRPV2, the level of expression of mRNA corresponding to the TRPV2gene can be detected utilizing commonly used molecular biologicalmethods, for example, Northern blotting, in situ hybridization, nucleaseprotection assays, RT-PCR (including real-time, quantitative PCR), highdensity arrays and other hybridization methods. Accordingly, in anotherembodiment, an assay capable of detecting the expression of one or morethan one TRPV2 gene in a biological sample is provided, which comprisescontacting a biological sample with an oligonucleotide capable ofhybridizing to a TRPV2 nucleic acid. The oligonucleotide is generallyfrom 10-20 nucleotides in length for PCR/primer extension experiments.Longer oligonucleotides of approximately 40-50 nucleotides are moreregularly utilized for in situ or blot hybridizations. Sequences evenlonger than 50 nucleotides can also be employed in the detection methodsof this invention. RNA can be isolated from the tissue sample by methodswell-known to those skilled in the art as described, for example, inAusubel et al., Current Protocols in Molecular Biology, John Wiley and.Sons, Inc. (1996). One preferred method for detecting the level of mRNAtranscribed from the TRPV2 genes is by RT-PCR. Details of RT-PCRtechniques are well known and also described, for example, in Pfeffer etal. Efficient one-tube RT-PCR amplification of rare transcripts usingshort sequence-specific reverse transcription primers. BioTechniques 18,204-206. (1995) and Giannoni et al. An improved reversetranscription-polymerase chain reaction method to study a lipoproteingene. Journal of Lipid Research 35 (2): 340 (1994).

Optionally, all or a portion of the TRPV2 nucleic acid sequence can beused to probe nucleic acid preparations from other species to determinethe presence of similar sequences. For example, all or a portion of theTRPV2 nucleic acid can be used as a probe to identify cDNA or genomicnucleic acid sequences from other species that are similar to the TRPV2sequence. Positive clones can be identified as those that hybridize tothe TRPV2 probe.

The invention also relates to isolated nucleic acid fragments. Isolatednucleic acids comprising fragments of SEQ ID NO: 1 are useful for avariety of purposes. For example, these sequences can be used asoligonucleotide probes for the detection of TRPV2 nucleic acids or forthe detection of sequences that flank TRPV2 nucleic acids. Thesesequences can be used as oligonucleotide primers for the amplificationof TRPV2 nucleic acids. For many methods, oligonucleotides of about16-25 nucleotides, or from about 26-35 nucleotides in length are useful,although longer oligonucleotides of greater than about 35 nucleotidescan also be utilized. Some oligonucleotides can be used as “primers” forthe synthesis of complimentary nucleic acid strands. For example, DNAoligonucleotide primers can hybridize to a complimentary nucleic acidsequence to prime the synthesis of a complimentary DNA strand inreactions using DNA polymerases. Oligonucleotides are also useful forhybridization in several methods of nucleic acid detection, for example,in Northern blotting or in situ hybridization. Fluorescently-labeledoligonucleotides, for example, Cy3-dCTP-labeled fluorescent cDNA probes,are particularly useful in hybridization methods using microarrays.Oligonucleotides can also be used for the preparation of chimericnucleic acids that encode a portion or all of the TRPV2 polypeptidefused to another polypeptide sequence, for example, one or more than onemotif or domain of the TRPV2 sequence recombined with one or more thanone motif or domain from one or more than one heterologous sequence. Insome embodiments, this can affect the activity of the TRPV2 channel.Oligonucleotides can be used in other methods; for example, RT-PCR usingSEQ ID: 5 and SEQ ID: 6 or SEQ ID: 7 and SEQ ID: 8 can be used toidentify TRPV2 expression. Oligonucleotides can further be used intechniques such as RNAi to reduce TRPV2 expression. In addition tonucleic acid sequences encoding TRPV2 polypeptides, the invention alsoincludes TRPV2 polypeptides, TRPV2 polypeptide variants, fragments ofTRPV2 polypeptides and. TRPV2 polypeptides having additional aminoacids.

TRPV2 polypeptide variants are polypeptides capable of TRPV2 activity inwhich substitutions have been made in the amino acid sequence from thesequence shown in SEQ ID NO: 2. These substitutions can be as a resultof naturally occurring mutations during DNA synthesis, transcription ortranslation, the use of amino acid analogs, chemical modifications orother molecular biology techniques well known in the art.

Fragments of TRPV2 polypeptides include polypeptides capable of TRPV2activity, which are shorter in length than the sequence shown in SEQ IDNO: 2. These fragments include any polypeptide capable of TRPV2activity, which are less than around 95 amino acids in length. Forexample, a fragment of TRPV2 can be from 65 to about 735 amino acids inlength and still be capable of TRPV2 activity. Fragments of TRPV2polypeptides may be created by naturally occurring mutations during DNAsynthesis, transcription or translation. Those skilled in the art willreadily recognize that the use of enzymes that cleave either DNA orproteins, chemical modifications or other molecular biology techniquescan be employed to create fragments of TRPV2 polypeptides.

TRPV2 polypeptides or TRPV2 polypeptide fragments can be generated usingany sort of synthetic or molecular biological technique. Standardsynthetic peptide techniques can be used to generate smaller TRPV2polypeptide fragments. Techniques for the synthesis of peptide fragmentsare well known and are described in, for example, Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al., J. Am. Chem. Soc., 85: 2149-2156 (1963), and.Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

TRPV2 polypeptides that have additional amino acids and are longer inlength than the sequence shown in SEQ ID NO: 2 are also envisionedwithin the scope of the invention. As described herein, the TRPV2polypeptide can also have additional amino acid residues at its aminoterminus, its carboxyl terminus or both. Such additional residues areuseful for a variety or purposes, including, for example,immunodetection, purification, cellular trafficking, enzymatic activity,etc. These proteins can include any polypeptide capable of TRPV2activity, which is greater than 10 amino acids in length. For example arecombinant TRPV2 can be from 1 to about 775 amino acids in length andstill be capable of TRPV2 activity. Recombinant TRPV2 polypeptides canbe created by naturally occurring mutations during DNA synthesis,transcription or translation, the use of enzymes that either ligate DNA,such as T4 ligase, or fuse proteins.

Additionally fragments of TRPV2 and TRPV2 polypeptides having additionalamino acids can comprise variant sequences in which substitutions havebeen made in the amino acid sequence from the sequence shown in SEQ IDNO: 2. These substitutions can be as a result of naturally occurringmutations during DNA synthesis, transcription or translation, the use ofamino acid analogs, chemical modifications or other molecular biologytechniques well known in the art.

A nucleic acid sequence can encode a TRPV2 fusion protein, which caninclude additional amino acid residues providing coordinates forbonding, such as ionic, covalent, hydrogen or Van der Waals orcombinations thereof with organic or inorganic compounds. Usefuladditional amino acid sequences include, for example, poly-histidineresidues useful for protein purification via Ni+-coupled residue,constant domains of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3), albumin, hemagluttinin (HA) or myc affinityepitope tags useful for the formation of immuno-complexes for detectionor purification (antibodies against these moieties can be obtainedcommercially), polypeptides useful for detection such as the greenfluorescent protein (GFP), enzymes, such as beta-galactosidase (β-Gal),glutathione S-transferase (GST), chloramphenicol acetyltransferase(CAT), luciferase, and alkaline phosphatase (A), signal sequences forprotein trafficking and protease cleavage sequences useful forseparating additional amino acid sequences from the TRPV2 sequence, ifdesired. Those skilled in the art will readily recognize that chemicalmodifications or other molecular biology techniques can be employed tocreate recombinant proteins or protein fusions. Fragments of TRPV2 andTRPV2 polypeptides having additional amino acids can comprise variantsequences in which substitutions have been made in the amino acidsequence as compared with the sequence shown in SEQ ID NO: 2. Thesesubstitutions can be as a result of naturally occurring mutations duringDNA synthesis, transcription or translation, the use of amino acidanalogs, other molecular biology techniques, or chemical modificationswell known in the art.

Due to the degeneracy of the genetic code, more than one codon can beused to encode a particular amino acid, and therefore, a TRPV2 aminoacid sequence (for example, SEQ ID NO: 2) can be encoded by any one of aplurality of nucleic acid sequences. Isolated nucleic acid includessequences wherein one or more than one codon in the sequence is replacedby one or more than one codon of a different sequence but that encodesthe same amino acid residue are herein referred to as “conservativecodon substitutions”. Therefore, the invention encompasses nucleic acidsequences encoding SEQ ID NO: 2, which have one or more than oneconservative codon substitution. One of skill in the art would be ableto determine a particular nucleic acid sequence having one or more thanone conservative codon substitution and encoding SEQ ID NO: 2, based onthe sequence information provided herein. Conservative codonsubstitutions can be made in the nucleic acid sequence encoding theTRPV2 polypeptide, for example, the codons TTT and TTC (collectivelyreferred to as TTT/C) can encode a Phe (phenylalanine) residue; othercodon substitutions are as follows: TTA/G and CTT/C/A/G: Leu; ATT/C:Ile; ATG: Met; GTT/C/A/G: Val; TCT/C/A/G: Ser; CCT/C/A/G: Pro;ACT/C/A/G: Thr; GCT/C/A/G: Ala; TAT/C: Tyr; CAT/C: His; CAA/G: Gln;AAT/C: Asn; AAA/G: Lys; GAT/C: Asp; GAA/G Glu; TGT/C: Cys; CGT/C/A/G:Arg; AGT/C: Ser; AGA/G; Arg; GGT/C/A/G:Gly. Conservative codonsubstitutions can be made at any position in the nucleic acid sequencethat encodes the TRPV2 polypeptide.

The isolated nucleic acids of the invention can also include nucleicacid sequences that encode the TRPV2 polypeptide having additional aminoacid residues. In some embodiments, the additional amino acids arepresent at the amino terminus, the carboxyl terminus, within the TRPV2sequence or combinations of these locations. TRPV2 polypeptides havingthese types of additional amino acid sequences can be referred to as“TRPV2 fusion proteins”. In some cases, it may be more appropriate torefer to them otherwise as “chimeric” or “tagged” TRPV2 proteins, or thelike, depending on the nature of the additional amino acid sequences.Nonetheless, one will be able to discern a TRPV2 polypeptide havingadditional amino acid sequences given the sequence information providedherein. The additional amino acid residues can be few in number, forexample, from one to about 20 additional amino acid residues, or longer,for example, greater than about 20 additional amino acid residues. Theadditional amino acid residues can serve one or more than one functionor purpose including, for example, serving as epitopes for protein(e.g., antibody) or small molecule binding; serving as tags forintracellular and extracellular trafficking; providing additionalenzymatic or other activity; or providing a detectable signal.

Recombinant techniques can be used for the expression of TRPV2 includingbut not limited to fragments of TRPV2, variants of TRPV2 and fusions ofTRPV2 with other proteins from host cells transformed with a TRPV2nucleic acid. These methods include, for example, in vitro recombinantDNA techniques and in vivo genetic recombination (for example, see thetechniques described in Sambrook et al., Molecular Cloning, A LaboratoryManual, 3rd Edition, Cold Spring Harbor Press, NY (2001); and Ausubel etal., eds., Short Protocols in Molecular Biology, 4th Edition, John Wiley& Sons, Inc., NY (1999)).

TRPV2 can be produced by introducing an expression vector encoding aTRPV2 polypeptide into a cell and culturing the cells to express thepolypeptide. When a purified TRPV2 polypeptide is desired, a step canalso be performed to isolate and, if desired, purify the TRPV2polypeptide.

In another embodiment, the invention provides a recombinant nucleic acidconstruct that includes all or a portion of the TRPV2 coding sequenceoperably linked to a regulatory sequence. These recombinant nucleic acidconstructs include recombinant expression vectors suitable forexpression of the TRPV2 nucleic acid in a host cell. Recombinantexpression vectors include one or more than one regulatory sequence thatcan be selected based on the type of host cells used for TRPV2expression, operably linked to a TRPV2 nucleic acid sequence. Regulatorysequences include promoters, enhancers and other expression controlelements, for example, poly (A)+ sequences. Regulatory sequences can bespecific for prokaryotic cells, for example, bacterial cells, such as E.coli, or for eukaryotic cells, such as yeast cells, insect cells ormammalian cells (for example, HEK, CHO or COS cells). Regulatorysequences can be located cis or trans relative to the TRPV2 nucleic acidsequence. Regulatory sequences can include constitutive expressionsequences that typically drive expression of the nucleic acid under awide variety of growth conditions and in a wide variety of host cells,tissue-specific regulatory sequences that drive expression in particularhost cells or tissues and inducible regulatory sequences that driveexpression in response to a secondary factor. The choice and design ofthe expression vector can depend on such factors as the particular hostcell utilized and the desired levels of polypeptide expression. Genesfacilitating selection of transformed cells encode proteins that conferresistance to antibiotics or other toxins, for example, ampicillin,neomycin, methotrexate or tetracycline, complement auxotrophicdeficiencies or supply critical nutrients not available from complexmedia.

Recombinant nucleic acid constructs used for expression of the TRPV2polypeptide can also include constructs that can be transcribed andtranslated in vitro, for example, constructs having a T7 promoterregulatory sequence.

Vectors suitable for the expression of TRPV2 are known in the art andcommercially available. Suitable vectors include, for example, pcDNA3.1,pCI-neo, pET-14b, pCDNA1 Amp and pVL1392, which are available fromNovagen and. Invitrogen and can be used for expression in E. coli, COScells and baculovirus infected insect cells, respectively.

In another embodiment, the invention provides a recombinant cell thatincludes a TRPV2 nucleic acid. Recombinant cells include those wherein anucleic acid sequence has been introduced. Typically, recombinant cellsare created by introducing a particular nucleic acid into cells usingmolecular biological techniques. However, recombinant cells also includecells that have been manipulated in other ways to promote the expressionof a desired nucleic acid sequence. For example, regions that areproximal to a target nucleic acid sequence can be altered to promoteexpression of the target nucleic acid, or genes that act to regulate theexpression of a target nucleic acid can be introduced into a cell.Recombinant cells, after periods of growth and division, may not beidentical to the starting parent cell; however, these cells are stillreferred to as recombinant cells and are included within the scope ofthe term as used herein.

Host cells suitable for harboring and providing the machinery for TRPV2expression include any prokaryotic or eukaryotic cells. Examples ofsuitable prokaryotic host cells are eubacteria, such as gram-negative orgram-positive organisms, for example, Enterobacteriaceae such asEscherichia, for example, E. coli, Enterobacter, Salmonella, forexample, Salmonella typhimurium, as well as Bacilli, such as B.subtilis, Pseudomonas, and Streptomyces. Many higher eukaryotic hostcells can be used, including insect cells, such as Drosophila S2 andSpodoptera Sf9 cells, mammalian cells, such as U937, THP-1, ChineseHamster Ovary (CHO) cells, canine kidney (COS) cells, canine kidney(MDCK) cells, human cervical carcinoma (HeLa) cells, and human embryonickidney (HEK) cells as well as plant cells.

Growth of the transformed host cells can occur under conditions that areknown in the art. The conditions will generally depend upon the hostcell and the type of vector used. Suitable induction conditions, such astemperature and chemicals, can be used and will depend on the type ofpromoter utilized. Examples of suitable media for the propagation ofprokaryotes include Luria Broth (LB), also known as Miller's L Broth.Examples of suitable media for the propagation of eukaryotes include,Minimal Essential Medium (MEM), RPMI-1640, Dulbecco's Modified Eagle'sMedium (DMEM) and F12 Medium.

Nucleic acids, including expression constructs, can be introduced intoprokaryotic or eukaryotic cells via conventional transformation ortransfection techniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing a foreign nucleic acid molecule (e.g., DNA)into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, virusmediated introduction, biolistics or electroporation. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook, etal. (supra) and other laboratory manuals.

Recombinant cells can be useful for the production of a TRPV2polypeptide for purification purposes or for functional studiesinvolving the TRPV2 polypeptide. For example, a recombinant TRPV2 cellcan be used to test a number of compounds for their ability to alter theactivity of the TRPV2 polypeptide. The recombinant TRPV2 cell can alsobe used to test how altering various properties of the TRPV2polypeptide, for example, altering the amino acid sequence of the TRPV2polypeptide, affects TRPV2 activity.

A variety of methods can be used for purification of the TRPV2polypeptide. For example, crude purification can be performed usingammonium sulfate precipitation, centrifugation or other knowntechniques. A higher degree of purification can be achieved by suitablechromatographic techniques, including, for example, anion exchange,cation exchange, high performance liquid chromatography (HPLC), gelfiltration, hydrophobic interaction chromatography and affinitychromatography, for example, immunoaffinity chromatography usingantibodies directed against the TRPV2 protein. If needed, steps forrefolding the TRPV2 proteins can be used to obtain the activeconformation of the protein when the protein is denatured duringintracellular synthesis, isolation or purification, such as resuspendingthe protein in 6M urea and dialyzing out the urea to facilitate proteinrefolding.

The invention also provides novel antibodies that selectively bind tothe proteins of the present invention as well as fragments thereof. Suchantibodies can be used to quantitatively or qualitatively detect theprotein or peptide molecules of the present invention. As used herein,an antibody selectively binds a target protein or polypeptide fragmentwhen an antibody binds the protein or polypeptide fragment and does notsignificantly bind to non-target proteins (i.e., the antibody does notbind non-TRPV2 related polypeptides or proteins).

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: antibodies are multi-subunit proteinsproduced by any vertebrate organism in response to an antigen challenge.The antibodies of the present invention include monoclonal antibodiesand polyclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)hd 2, Fc, and. Fv fragments.Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989). In general, to generateantibodies, an isolated peptide is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit or mouse.Either the full-length protein, an antigenic peptide fragment, or afusion protein can be used.

Monoclonal antibodies can be produced by hybridomas, which areimmortalized cell lines capable of secreting a specific monoclonalantibody. The immortalized cell lines can be created in vitro by fusingtwo different cell types, usually lymphocytes, one of which is a tumorcell.

Monoclonal antibodies can be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines inculture. These include, for example, the hybridoma technique (Kohler andMilstein, Nature, 256:495-497, 1975); the human B-cell hybridomatechnique (Kosbor et al., Immunology Today, 4:72, 1983); and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies can be ofany immunoglobulin class, including IgG, IgM, IgE, IgA, IgD or anysubclass thereof The hybridoma producing the monoclonal antibody of thisinvention can be cultivated in vitro or in vivo.

Antibodies can be prepared from regions or discrete fragments of theprotein containing the desired amino acid sequence. Antibodies can beprepared from any region of the peptide as described herein. However,regions that include those involved in function/activity and/orprotein/binding partner interaction are preferred. An antigenic fragmentwill typically comprise at least 10 contiguous amino acid residues. Theantigenic peptide can comprise, however, at least 12, 14, 20 or moreamino acid residues. Such fragments can be selected on a physicalproperty, such as fragments corresponding to regions that are located onthe surface of the protein (e.g., hydrophilic regions) or can beselected based on sequence uniqueness or based on the position ofparticular amino acid residue(s) or amino acid residue variants of thepolypeptides provided by the present invention.

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material isluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 125 I, 131 I, 35 S or 3 H.

Immunoprecipitation of Ab2 using the antibodies of the present inventionfor detection of proteins interacting with TRPV2 is also envisionedwithin the scope of the present invention. Immunoprecipitation can alsobe used, for example, to discover new disease targets. The TRPV2antibody can be linked to any immobilizing surface, such as ProteinG-sepharose or Protein A-sepharose for the purpose of TRPV2purification, to identify interacting proteins and potential drugtargets. Additionally, the process can be used to determine themolecular weight and isoelectric point of immunoprecipitated proteins byone-dimensional or two-dimensional SDS-PAGE or LC MS/MS.Immunoprecipitation can be used to verify that an antigen of interest issynthesized by a specific tissue (i.e., that a labeled protein can beidentified in tissues or cells cultured with labeled precursors). Thisprocedure can be used to determine whether a protein containscarbohydrate residues by evaluating whether immunoprecipitated antigenfrom cells cultured with labeled monosaccharides is labeled.Characterization of the type of covalent modification, such asphosphorylation or glycosylation wherein the carbohydrates present onglycoproteins, can be performed by evaluating the incorporation ofdifferent labeled monosaccharides into immunoprecipitated proteinsduring cell culture and testing whether inhibitors of glycosylationalter the molecular weight of the immunoprecipitated protein.Immunoprecipitation can be used to determine precursor-productrelationships by performing pulse-chase labeling followed byimmunoprecipitation. Immunoprecipitation can be used to quantifysynthesis rates of proteins in culture by determining the quantity ofimmunoprecipitated, labeled protein. Methods and reagents forimmunoprecipitation are well known in the art and can be obtained fromeBioscience, San Diego, Calif. or Upstate USA, Inc. Charlottesville, Va.The invention includes, antibodies useful for immunohistochemistry (IHC)and western blotting applications, for example, human TRPV2-specificantibodies against different regions within the canine TRPV2 protein.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology and recombinant DNA are used. Thesetechniques are well-known and are explained in, for example, CurrentProtocols in Molecular Biology, Vols. I, II, and III, F. M. Ausubel, ed.(1997); and Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold. Spring Harbor, N.Y. (2001).The compositions, kits, and methods of the invention have the followinguses, among others: 1) selecting a composition or therapy forinhibiting, modulating, or activating TRPV2 in a patient; 2) assessingthe efficacy of one or more than one test compound for inhibiting,modulating, or activating TRPV2 in a patient; 3) assessing the efficacyof a therapy for inhibiting, modulating, or activating TRPV2 in apatient; 4) treating a patient afflicted with certain types of pain orpainful conditions, including but not limited to neuropathic pain andinflammatory pain.

These types of compounds can be identified using a system that includesa TRPV2 polypeptide or a TRPV2 nucleic acid. Compounds can be testeddirectly in vivo, for example, in a rat, mouse, canine, or in modelsystems thereof or directly in bacterial cultures or in eukaryotic cellcultures. These methods comprise assaying for the ability of variouscompounds to increase or decrease the expression of the TRPV2 protein orthe activity of the TRPV2 protein as shown in Example 1. The compoundscreening and identification methods can be performed using conventionallaboratory formats or in assays adapted for high throughput.

Candidate compounds encompass numerous chemical classes, including butnot limited to, small organic or inorganic compounds, natural orsynthetic molecules, such as antibodies, proteins or fragments thereof,antisense nucleotides, interfering RNA (iRNA) and ribozymes. Preferably,the candidate compounds are small organic compounds (i.e., those havinga molecular weight of more than 100 Daltons yet less than about 1000Daltons). Candidate compounds comprise functional chemical groupsnecessary for structural interactions with polypeptides and can includeat least an amine, carbonyl, hydroxyl or carboxyl group, two of thefunctional chemical groups or three or more of the functional chemicalgroups. The candidate compounds can comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one of the above identified functional groups.Candidate compounds also can be biomolecules, such as peptides,saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines,derivatives or structural analogs of the above or combinations thereofand the like. Where the compound is a nucleic acid, the compoundtypically is a DNA or RNA molecule, although modified nucleic acidshaving non-natural bonds or subunits are also contemplated. Modifiednucleic acids can comprise nucleoside analogs, nucleotide analogs,non-nucleoside analogs, non-nucleotide analogs and others.

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random peptides and the like. Candidatecompounds can also be obtained using any of the numerous approaches incombinatorial library methods known in the art, including biologicallibrary methods; spatially addressable, parallel solid phase or solutionphase library methods, synthetic library methods requiringdeconvolution, the “one-bead one-compound” library method, and syntheticlibrary methods using affinity chromatography selection (Lam,Anti-Cancer Drug Design, 1997, 12:145). Alternatively, libraries ofnatural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural andsynthetically produced libraries and compounds can be readily modifiedthrough conventional chemical, physical and biochemical means.

Further, known pharmacological agents can be subjected to directed orrandom chemical modifications, such as acylation, alkylation,esterification, amidation, etc. to produce structural analogs of theagents. Candidate compounds can be selected randomly or can be based onexisting compounds that bind to and/or modulate the function of TRPV2activity. Therefore, a source of candidate agents is one or more thanone library of molecules based on one or more than one known compoundthat increases or decreases TRPV2 protein expression and/or G-proteincoupled receptor activity in which the structure of the compound ischanged at one or more than one position of the molecule to contain moreor fewer chemical moieties or different chemical moieties. Thestructural changes made to the molecules in creating the libraries ofanalog activators/inhibitors can be directed, random, or a combinationof both directed and random substitutions and/or additions. One ofordinary skill in the art in the preparation of combinatorial librariescan readily prepare such libraries based on the existing compounds.

Assays for the identification of TRPV2 modulators, such as antagonistsand agonists, can be carried out manually or using an automated system.Automated systems are preferred if high throughput screenings areperformed. For example, one type of automated system utilizes multi-wellculture plates, for example, 96-well, 384-well or 1536-well cultureplates, wherein each well can contain cells that express TRPV2endogenously, recombinant cells having a nucleic acid encoding the TRPV2protein or the purified TRPV2 protein.

The purified TRPV2 protein, antibodies against TRPV2 polypeptidesdisclosed herein and the various constructs used to express TRPV2 can beused in studies for understanding the mechanism of action of anidentified TRPV2 modulator such as an agonist or antagonist as well asunderstanding the function of the protein.

After a compound has been identified that meets the desired criteria formodulating TRPV2 expression or activity, the compound can then beadministered to a live subject, such as a living cell or other organism,or to cultured cells or tissues expressing TRPV2 to assess the efficacy.This can be useful to establish toxicity and other pharmacologicalparameters of the compound important for determining dosing regimens.For example, after a compound is identified using an ex vivo systemcontaining a TRPV2 polypeptide, the compound can be administered to aculture of cells or an organism to examine various pharmacologicalaspects of the compound. Additionally, animal models can be used inconjunction with the nucleotides, antibodies and kits described hereinto assess the toxicity of a compound. The TRPV2 systems as describedherein are particularly advantageous for identifying and establishingdosing regimens in humans, because animals, particularly large specie,such as canines and primates, can be used, which are closer in weight tohumans, compared with rats or mice, and therefore provide a moresuitable animal model for estimating human dosing.

In another embodiment, the invention provides a method of identifying acompound useful for treating certain types of pain or painfulconditions, including but not limited to neuropathic pain andinflammatory pain, comprising the steps of: (a) contacting a sampleexpressing TRPV2 with a test compound; and (b) determining whether thetest compound increases or decreases the expression or activity ofTRPV2. In some embodiments, the method further comprises the steps of:(a) administering the test compound to an animal; and (b) determiningthe extent to which the test compound affects the nociceptive status ofthe animal.

Various animal models of pain exist, for example, the spinal nerveligation (SNL) model of nerve injury, which is a neuropathic pain modelin rats developed by Kim and Chung (Pain, 50:355-363, 1992). Othersuitable animal models of pain can be utilized in connection with theteachings herein. Commonly studied rodent models of neuropathic paininclude the chronic constriction injury (CCI) or Bennett model; neuromaor axotomy model; and the partial sciatic transection or Seltzer model(Shir et al., Neurosci. Lett., 115:62-67, 1990). Exemplary neuropathicpain models include several traumatic nerve injury preparations (Bennettet al., Pain 33: 87-107, 1988; Decosterd et al., Pain 87: 149-58, 2000;Kim et al., Pain 50: 355-363, 1992; Shir et al., Neurosci Lett 115:62-7, 1990), neuroinflammation models (Chacur et al., Pain 94: 231-44,2001; Milligan et al., Brain Res 861: 105-16, 2000) diabetic neuropathy(Calcutt et al., Br J Pharmacol 122: 1478-82, 1997), virus-inducedneuropathy (Fleetwood-Walker et al., J Gen Virol 80: 2433-6, 1999),vincristine neuropathy (Aley e t al., Neuroscience 73: 259-65, 1996;Nozaki-Taguchi et al., Pain 93: 69-76, 2001), and paclitaxel neuropathy(Cavaletti et al., Exp Neurol 133: 64-72, 1995), as well as acutenociceptive tests models and inflammatory models, such as those in whichinflammation is produced by an injection of carrageenan, completeFreund's adjuvant (CFA), zymosan or capsaicin (Brennan, T. J. et al.Pain 64:493, 1996; D′Amour, F. E. and Smith, D. L. J Pharmacol 72:74-79, 1941; Eddy, N. B. et al. J Pharmacol Exp Ther 98:121, 1950;Haffner, F. Dtsch Med Wochenschr 55:731, 1929; Hargreaves, K. et al.Pain 32: 77-88, 1988; Hunskaar, S. et al. J Neurosci Meth 14:69, 1985;Randall, L. O. and. Selitto, J. J. Arch. Int. Pharmacodyn 111: 409-419,1957; Siegmund, E. et al. Proc Soc Exp Bio Med 95:729, 1957). In anotheraspect, the animal model involves a dog, or a primate, for example, acanine or a human.

Therapeutic efficacy and toxicity can be determined by standardpharmaceutical procedures in cell cultures or experimental animals bycalculating, for example, the ED₅₀ (the dose therapeutically effectivein 50% of the population or the dose that produces 50% of the maximaldesired effect) and the LD₅₀ (the dose lethal to 50% of the population).The dose ratio between toxic and therapeutic effects is the therapeuticindex, and can be expressed as the ratio, LD₅₀/ED₅₀. The data obtainedfrom cell culture assays using TRPV2 polypeptide, cells expressing TRPV2and/or animal studies, such as canine or primate studies, are used informulating a range of dosage for human use. The dosage contained insuch compositions preferably gives rise to a range of circulatingconcentrations that include and/or exceed the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient and the route ofadministration.

The exact dosage will be determined by the one administering the dose,in light of factors related to the subject requiring treatment. Dosageand administration are adjusted to provide sufficient levels of theactive agent or to maintain the desired effect, for example, control ofpain levels. Factors that can be taken into account include the generalhealth, age, weight and gender of the subject, diet, time and frequencyof administration, drug combination(s), reaction sensitivities andtolerance/response to therapy.

The pharmaceutical compositions containing a compound that has beenidentified as modulating TRPV2 expression or activity can beadministered by any number of routes, including, but not limited to,oral, intravenous, intramuscular, intraarticular, intraarterial,intramedullary, intrathecal, epidural, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,inhalational, intraocular, intra-aural or rectal means.

In addition to the active ingredients, these pharmaceutical compositionscan contain suitable, pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically or thatfacilitate absorption or distribution of the active compounds. Furtherdetails on techniques for formulation and administration can be found inRemington's Pharmaceutical Sciences, 20^(th) edition, Maack PublishingCo., Easton, Pa. (2000). Pharmaceutical compositions for oraladministration can be formulated using pharmaceutically acceptablecarriers well known in the art in dosages suitable for oraladministration. Such carriers enable the pharmaceutical compositions tobe formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for ingestion by the patient.

Example 1

The present invention is directed to an isolation of a cDNA from caninedorsal root ganglion (DRG) tissue (SEQ ID NO: 1) encoding a full lengthTRPV2 (SEQ ID NO: 2), its preparation, characterization and functionalexpression, the establishment of a high throughput assay for testing theability of compounds to alter the function of TRPV2 and the evaluationfor the potential to decrease pain in animals and humans. In order toclone TRPV2 from canine, messenger RNA was first isolated from canineDRG, a reverse transcriptional PCR (RT-PCR) and a Rapid Amplification ofcDNA End (RACE-PCR) technique were used. The cloning result revealedthat the open reading frame cTRPV2 contains 2298 by encoding apolypeptide of 765 amino acids and having a calculated molecular mass of85.4 kDa. A Kyte-Doolitle hydrophilicity analysis of primary sequencepredicts the presence of six putative hydrophobic domains clustered nearthe carboxyl terminus, which may form a pore of the ion channel. Thenucleotide sequence of canine TRPV2 is 83%, 82% and 86% identical to themouse, rat and human TRPV2, respectively. (See FIG. 4).

A. Isolation of Poly(A⁺) RNA

As a first step in the cloning of cTRPV2, poly(A⁺) RNA was isolated from100 μg of total RNA from canine DRG (Custom made by AnalyticalBiological Service Inc. DE) using an Oligotex™ spin column (Qiagen Inc.,CA). Briefly, 150 μl of RNase-free water, 250 μl of buffer OBB,containing 20 mM Tris (pH=7.5), 1M NaCl, 2 mM EDTA and 0.2% SDS, and 15μl of a suspension of Oligotex beads were added to 100 μl of total RNAsolution (1 μg/μl). The RNA/Oligotex bead mixture was then heated at 70°C. for 3 min, to disrupt any secondary structure of the RNA, followed byincubation at room temperature for 10 min. The poly (A⁺) RNA/Oligotexparticle complex was centrifuged and washed twice with 400 μl of bufferOW2, containing 10 mM Tris (pH=7.5), 150 mM NaCl and 1 mM EDTA, and thentransferred to a spin column for the elution step. The poly(A⁺) RNA waseluted from Oligotex bead using 200 μl of prewarmed (70° C.) buffer OEB,containing 5 mM Tris (pH=7.5). Finally, canine DRG poly(A⁺) RNA wasprecipitated by ethanol in the presence of 20 μg of glycogen and 150 mMsodium acetate and resuspended in 10 μl of RNase-free water.

B. Synthesis of Double-stranded cDNA

4 μl (1 μg) of canine DRG poly(A⁺) RNA and 1 μl of cDNA synthesisprimer, a 52-mer oligo with sequence of5′-TTCTAGAATTCAGCGGCCGC(T)₃₀N⁻¹N-3′, N⁻¹=G, A or C; and N=G, A, C or T(Clontech, CA SEQ ID NO: 12), were mixed, incubated at 70° C. for 2 minand then cooled on ice for 2 min. The first strand cDNA synthesis(reverse transcription) was performed at 42° C. for 1 hour using 20units of AMV reverse transcriptase in the presence of 1 mM dNTP mixtureand first strand synthesis buffer, containing 50 mM Tris (pH=8.5), 8 mMMgCl₂, 30 mM KCl and 1 mM DTT, in 10 μl. The second strand cDNAsynthesis was performed by adding an enzyme cocktail, consisting of 24units of E. coli DNA polymerase I, 5 units of E. coli DNA ligase I unitof E. coli RNasc H, 0.25 mM of dNTP mixture (0.25 mM of each dATP, dCTP,dGTP, and dTTP) and second strand buffer, containing 100 mM KCl, 10 mMammonium sulfate, 5 mM MgCl₂, 0.15 mM (β-NAD, 20 mM Tris (pH=7.5) and 50μM bovine serum albumin, in 80 μl. The reaction was first carried out at16° C. for 90 min followed by addition of 20 units of T4 DNA polymerase,with continued incubation at the same temperature for 45 min. Thereaction was terminated by adding 10 mM EDTA and 8 μg of glycogen.Phenol and chloroform extractions were performed, followed by ethanolprecipitation. Double-stranded cDNA was then suspended in 200 μl of TEbuffer and stored at −20° C.

C. PCR Amplification of Near Carboxyl Terminus of cTRPV2

A TRPV2 cDNA fragment was successfully amplified by PCR using twoprimers designated as forward primer dv2-6 (5′-TGATGATCGCAGACAACTCAGCCGAGAACA-3′) (SEQ ID NO: 5), corresponding to nucleotides 761-790 ofSEQ ID NO 1, and reverse primer dv2-8 (5′-GGACAGCTGGGCCTGATGGCTCTTCA-3′) (SEQ ID NO: 6), corresponding to nucleotides 2175-2200 of SEQID NO 1. The PCR reaction was performed in a final volume of 50containing 5 μl of canine DRG double-stranded cDNA, 5 μl of 10× reactionbuffer (provided with Advantage2 DNA polymerase), 200 μM dNTPs, 200 nMforward primer dv2-6, 200 nM reverse primer dv2-8 and 1 μl of 50×Advantage™2 DNA polymerase mixture (Clontech, Calif.). PCR was performedby an initial denaturing step at 94° C. for 1 min, followed by 30 cyclesof: (a) denaturing at 94° C. for 30 sec, (b) annealing at 55° C. for 30sec and (c) extension at 72° C. for 60 sec. Agarose gel electrophoresiswas performed, which revealed that the PCR product was approximately1.44 kb. After PCR, the 1.44 kb PCR fragment was purified and subclonedinto pCRII TA cloning vector (Invitrogen, Calif.) following the vendor'sprotocol. Six independent clones were picked and subjected to DNAsequencing analysis. The sequence results revealed that the PCRamplified fragment was 82%, 81% and 87% identical to the comparableregion, i.e., near carboxyl termini of mouse, rat and human TRPV2,respectively.

D. RACE-PCR of 5′ and 3′ Ends of cTRPV2 Sequence

To obtain the complete 5′ and 3′ cDNA sequences of the cTRPV2 gene,RACE-PCR technology was performed. First, both 5′- and 3′-RACE-ReadycDNAs were synthesized separately with SMART™ RACE DNA Amplification Kit(BD Clontech, CA), according to the manufacturer's instructions. Toprepare cDNA for 5′ RACE, in one 0.5 ml tube, 3 μl of dog DRG poly(A⁺)RNA obtained in step A above. was mixed with 1 μl of 5′-CDS primer and 1μl of SMART II A oligo. To prepare cDNA for 3′ RACE, 3 μl of canine DRGpoly(A⁺) RNA was mixed with 1 μl 3′-CDS primer and 1 μl RNase free waterin another 0.5 ml tube and then incubated at 70° C. for 2 min, followedby cooling on ice for 2 min. Next, 2 μl of 5× First Strand buffer, 1 μl20 mM DTT, 1 μl of 10 mM dNTP mix and 1 μl PowerScript ReverseTranscriptase were added to each tube, and synthesis was performed at42° C. for 90 min. The reactions were stopped by adding 200 μl of TEbuffer and heating the sample to 72° C. for 7 min. The reaction productswere stored at −20° C. All of the oligos, 5′-CDS, 3 ′-CDS and SMARTIIwere provided by Clontech.

For RACE-PCR, two primers were synthesized based on the above 1.44 kbcDNA sequence the cTRPV2 cDNA. The forward primer for 3′ RACE-PCR wasdv2-6; the reverse primer for 5′-RACE-PCR was named dv2-2(5′-GATTTTGCCCTCCTTGGCAGCCAG-3′) (SEQ ID NO: 7), which corresponds tonucleotides 901-924 of SEQ ID NO: 1. Both 5′ and 3′-RACE PCRs wereperformed in a final volume of 50 μl containing 2.5 μl of cDNA template(either 5′- or 3′-RACE-Ready cDNA, as described above), 5 pl of 10×reaction buffer, 200 μM dNTPs, 200 nM Universal Primer Mix (UPM)(Clontech), 200 nM cTRPV2 specific primer (dv2-2 for 5′-RACE PCR ordv2-2 for 3′-RACE PCR) and 1 μl of 50× Advantage™-HF2 DNA polymerasemixture (Clontech). The thermal cycler parameter for the RACE-PCR was 25cycles of 94° C. for 30 sec and 68° C. for 3 min. After the RACE-PCRreaction, the products were diluted 50-fold and used for the next roundnested PCR. For nested PCR, two oligos were synthesized. They werereverse primer dv2-9 (5′-TTGCCAGAGTTCCGGTCGATCTGAAGCA-3′) (SEQ ID NO:8), used for the 5′-nested-PCR and forward primer dv2-10(5′-TCCAAGACCAGCTGCGCTTCCGC GGCGT-3′) (SEQ ID NO: 9) used for the 3′nested-PCR. Both 5′ and 3′-nested-PCRs were performed in a final volumeof 50 μl containing 5 μl of a 1:50 dilution of the previous RACE-PCRproduct, 5 μl of 10× reaction buffer, 200 μM dNTPs, 200 nM UniversalPrimer Mix (UPM) (Clontech), 200 nM cTRPV2 specific primer (dv2-9 for 5′nested-PCR or dv2-10 for 3′ nested-PCR) and 1 μl of 50× Advantage2™DNApolymerase mixture (Clontech). The thermal cycler parameters for theRACE-PCR were 20 cycles of 94° C. for 30 sec and 68° C. for 3 min. Thenested-PCR products were purified and directly subcloned into pCRII TAcloning vector (Invitrogen). Eight to eighteen independent clones fromeither 5′-RACE/nested or 3′-RACE/nested PCR were picked and subjected toDNA sequencing analysis.

E. Sequence of Full-length cTRPV2 cDNA:

The sequence of the full-length canine TRPV2 cDNA was confirmed bysynthesizing PCR primers based on the sequence of the 5′ and 3′ endsobtained by RACE-PCR. Full length cTRPV2 cDNA was amplified from canineDRG double-stranded cDNAs prepared in step B above. by high-fidelity DNApolymerase using two sets of primers. The first primer set was forwardprimer dv2-1 (5′-ATGGCCTCGC CCTCCAGCTC TCCCA-3′) (SEQ ID NO: 10) andreverse primer dv2-2 for amplification of a 924 by cDNA fragmentencoding cTRPV2 amino terminal 308 residues; the second primer set wasforward primer dv2-6 and reverse primer dv2-11 (5′-TTAGTGGGACTTCAGGAGCTGGAGG GGC-3′) (SEQ ID NO: 11) for amplification of a 1.54 kb cDNAfragment encoding cTRPV2 carboxyl terminal 512 residues. The PCR wasperformed in a final volume of 50 μl containing 5 μl of above canine DRGdouble-stranded cDNAs (prepared as above), 5 μl of 10 × reaction buffer,200 μM dNTP, 200 nM forward primer (either dv2-1 or dv2-6) and reverseprimer (either dv2-2 or d2-11), and 1 μl of 50 × Advantage™-HF2 DNApolymerase mixture (Clontech, CA). The PCR was performed by an initialdenaturing step at 94° C. for 2 min, followed by 35 cycles of denaturingat 94 ° C. for 30 sec and annealing and extension at 70° C. for 5 min.After PCR, the 2.3 kb PCR fragment was purified and subcloned into pCRIITA cloning vector, following the same cloning protocol as in step Cabove. Several independent clones were picked and subjected to DNAsequencing analysis. The clones NQC881 and NQC918 were used for furthersubcloning and studying. The sequence results revealed that the openreading frame nucleic acid sequence of cTRPV2 cDNA SEQ ID NO 1 was 83%,82% and 86% identical to the cDNA sequences of mouse (Accession number:BC005415), rat (Accession number: AF129113) and human (Accession number:AF129112; SEQ ID NO: 3) TRPV2, respectively. (See FIG. 4).

F. Sequence Analysis

5′- and 3′-RACE-PCR facilitated the determination of the 68 by sequenceof the 5′ untranslated region; 3′-RACE-PCR allowed for the determinationof the 431 by sequence of the 3′ untranslated region, including the37-mer poly(A) tail. No in-frame stop codon was identified in either 5′or 3′ untranslated region.

The predicted cTRPV2 open reading frame consists of a 2298 by sequencethat is predicted to encode a polypeptide of 765 amino acids (SEQ ID NO:2), having a calculated molecular mass of 85.4 kDa. A Kyte-Doolitlehydrophilicity analysis (not shown) of primary sequence predicts thepresence of six putative hydrophobic (or transmembrane) domainsclustered at the carboxyl half of the molecule and believed to form thepore of the ion channel.

The cTRPV2 amino acid sequence was aligned with the human (GenBank Acc.No.: AF129112; SEQ ID NO: 4), rat (GenBank Acc. No.: AF129113), andmouse (GenBank Acc. No.: BC005415) sequences, which revealed 84%, 81%and 83% sequence identity, respectively (FIG. 4), using the Gap programfrom Seqweb version 2 of Accelrys. The Gap program uses the algorithm ofNeedleman and Wunsch (J Mol. Biol., 48:443 (1970)) to find the alignmentof two complete sequences, by maximizing the number matches andminimizing the number of gaps. A search of canine genomic sequence withTRPV2 cDNA revealed that cTRPV2 is located at chromosome 5, spanning17435 by with a total of 14 exons.

Example 2 Recombinant Expression of cTRPV2

A. Cloning of Canine TRPV2 into a Mammalian Expression Vector

For expression of cTRPV2 in mammalian cell lines, the full-length cDNAof cTRPV2 was subcloned into pcDNA3.1 (neo) by performing a three-wayligation. First, the clone NQC881 was digested with NotI and XhoI toyield a 0.9 kb 5′ fragment, and the clone NQC918 was digested with XhoIand EcoRI to yield a 1.45 kb 3′ fragment. The 0.9 kb 5′ and 1.45 kb 3′cTRPV2 fragments were purified and ligated with pcDNA3.1 that waspredigested with NotI and EocRI, creating the expression constructcTRPV2/pcDNA3.1.

B. Transient Transfection of Expression Construct into Mammalian Cells

The expression construct, cTRPV2/pcDNA3.1 was then transfected intohuman embryonic kidney cells (HEK293, ATCC CRL-1573) with FuGene6transfection reagent (Roche, Indianapolis, Ind.), according to thevendor's protocol. TRPV2-expressing HEK293 cells were cultured in DMEMsupplemented with 10% fetal bovine serum, 100 units/ml penicillin and100 μg/ml streptomycin at 37° C. with 5% CO₂, and were analyzed using acalcium influx assay 48-72 hr. post-transfection.

Example 3 Calcium Influx Functional Assay of cTRPV2

To demonstrate the functionality of the cTRPV2 gene, HEK293 cells weretransiently transfected with plasmids encoding either cTRPV2 or vectorcontrol and then evaluated in a FLIPR assay to measure changes inintracellular calcium levels of the cells in each well upon addition ofagonist. Transiently transfected cells were seeded in 384-well plates ata density of 1×10⁴ cells/well and incubated overnight at 37° C. Thefollowing day, the cells were loaded with buffer and calcium dye, asrecommended by the vendor (Molecular Devices, Sunnyvale, Calif.), in afinal volume of 40μl and incubated for 30 minutes at room temperature.The fluorescence intensity was measured by FLIPR before, during andafter the addition of Δ⁹-THC, which was added to the cells at a finalconcentration of 100 μM. The results are shown in FIG. 6.

Example 4 A Screening Assay for a Desensitizer or Inactivator of a TRPV2Channel

Generally, upon prolonged exposure of an ion channel to an activatingstimulus (e.g., an agonist) or in response to a direct desensitizing orinactivating stimulus, the channel may assume alternate conformationsthat are variably less activatable in response to an activatingstimulus. These less activatable or inactivatable conformations may bereferred to functionally as being desensitized or inactivated, andcompounds that produce these states as being desensitizers orinactivators, respectively. Such conformations may be induced orstabilized by or in the presence of these so-called desensitizers orinactivators, and may arise by the preferential action of thedesensitizer or inactivator upon an open or upon a closed channel. Inaddition, such conformations may be reversible, across variable timecourses and conditions, or may be irreversible, pending de novosynthesis of nascent channels. Compounds that are identified asdesensitizers or inactivators, either being reversible or irreversible,may be useful in the treatment of certain conditions, including painconditions, in which decreased TRPV2 activity would be therapeutic.

Therefore, in another embodiment, the invention provides a method ofidentifying reversible and irreversible desensitizers or inactivators ofcTRPV2 activity. The first method is designed to identify compounds thatinduce and/or stabilize the channel in a desensitized or inactivatedstate from a closed state and comprises the steps of: (a) providing arecombinant cell comprising a nucleic acid encoding a cTRPV2 protein,(b) contacting the recombinant cell at a temperature above the thresholdfor activation (typically above about 52° C.) with a test compound forvarying lengths of time, (c) contacting the recombinant cell withagonist for activation in the presence of a test compound for varyinglengths of time, (d) extensively washing out the test compound and (e)at varying time points, determining the extent to which the testcompound diminishes TRPV2 activity in response to a subsequent exposureto a TRPV2-activating stimulus. The second method is designed toidentify compounds that induce and/or stabilize the channel in adesensitized or inactivated state from an open state and comprisesEITHER the steps of: (a) providing a recombinant cell comprising anucleic acid encoding a cTRPV2 protein, (b) contacting the recombinantcell at a temperature above the threshold for activation (typicallyabove about 52° C.) or with a TRPV2 agonist, (c) contacting therecombinant cell with a test compound for varying lengths of time, (d)extensively washing out the test compound and agonist and (e) at varyingtime points, determining the extent to which the test compounddiminishes TRPV2 activity in response to a subsequent exposure to aTRPV2-activating stimulus OR the steps of: (a) providing a recombinantcell comprising a nucleic acid encoding a cTRPV2 protein, (b) incubatingthe recombinant cell at a temperature below the threshold foractivation, (c) contacting the recombinant cell with a test compound forvarying lengths of time, (d) extensively washing out the test compoundand (e) at varying time points, determining the extent to which the testcompound diminishes TRPV2 activity in response to a subsequent exposureto a TRPV2-activating stimulus.

1. A substantially purified polypeptide capable of TRPV2 activity andhaving at least 90% sequence identity to SEQ ID NO:
 2. 2. Thesubstantially purified polypeptide of claim 1 comprising SEQ ID NO: 2.